The invention relates to a method of measuring the luminescence emitted in a luminescent assay, which makes it possible to correct certain perturbations due to the measuring medium.
At the present time, immunoassays are widely used for the qualitative and quantitative analysis of compounds in biological fluids.
Among the techniques in existence, fluorimetric assays have become increasingly important.
In fact, they have a number of advantages, including the sensitivity and rapidity of the measurement, the stability and safety of the reagents labeled with fluorescent compounds, and the relatively low cost.
It is known that detection methods which use fluorescence are intrinsically very sensitive and could permit lower detection limits than those achieved by immunoassays which use radiolabeled reagents, in particular if modulatable laser light sources are used (I. Wieder, Immunofluorescence and related staining techniques, 1978, Elsevier).
Numerous fluorescent molecules usable as tracers in assays of this type have previously been described; among these, rare earth complexes, which possess valuable properties, may be mentioned in particular.
xe2x80x9cTracerxe2x80x9d is understood as meaning either a luminescent molecule emitting a direct luminescence, or a luminescent molecule capable of inducing a luminescent emission, it being possible for said molecule to be coupled with one of the reagents of the assay, and the emission of a direct or induced luminescence enabling the target analyte to be detected and/or determined.
The use of particular complexes, rare earth cryptates, is described for example in European patent applications 0 321 353, 0 180 492 and 0 232 348 or international patent application WO 90/04791.
These rare earth cryptates have the advantage of being very stable in a saline protein medium, this property being particularly important in the case of homogeneous immunoassays.
The sensitivity of the measurement is nevertheless limited by different interference parameters, among which the following may be mentioned:
the spectroscopic properties of the medium, and in particular its intrinsic fluorescence, which is due especially to the interference emissions of the molecules present in the measuring medium and capable of being excited and of emitting at wavelengths close to those of the fluorescent tracer and/or with strong intensities; its absorption, which results in a loss of exciting light; and its light diffusion properties when the measuring medium is not clear;
the quenching of the emitted fluorescence by inhibitors present in the medium; and
the composition of the equipment, and especially the interference reflections caused by the equipment.
Together these interferences considerably affect the sensitivity and reproducibility of the measurement.
Some of these problems have already been solved by a variety of techniques.
In particular, the time-resolved methods of measuring fluorescence enable the problem of interference emissions (background) to be partially overcome. The principle of these methods consists in measuring the fluorescence emitted by a tracer molecule having a relatively long emission lifetime, the measurement being delayed in time beyond the emission lifetime of the other molecules present.
It is necessary in this case to use fluorescent tracer molecules with a relatively long lifetime, such as rare earth chelates and cryptates.
Nevertheless, the problem of the limitations due to the spectroscopic properties of the medium, and in particular to its absorption, has not been solved satisfactorily.
In fact, none of the proposed techniques for avoiding the filtering effect of the medium makes it possible to carry out the measurement easily and inexpensively and at the same time to obtain a high sensitivity and a very good reproducibility in the measurement.
In particular, the solution which consists in greatly diluting the sample detracts from the sensitivity of the detection.
Furthermore, the use of a double exciting beam system involves the use of expensive equipment and special measuring cuvettes which are difficult to standardize. Moreover, systematic measurement of the absorption of the medium prior to measurement of the fluorescence of the sample complicates the assay method.
European patent application 355 849 describes a method and an automatic apparatus for checking the reliability of the fluorescent measurement of a sample and for correcting this measurement on the basis of an internal reference. To do this, measurement of the test sample has to be preceded by a measurement with 2 reference samples, one being a xe2x80x9cblankxe2x80x9d containing only the measuring medium, and the other containing the measuring medium and the fluorescent marker.
European patent application 91 126 relates to a fluorimeter which makes it possible to measure, in parallel with the fluorescence of the sample, the transmission of the sample and the fluctuations in excitation energy, in order to correct the measured fluorescence. This system requires a particular measuring cell which allows the exciting beam to pass through, since the transmission measurement has to be made in line with the exciting beam.
Other systems employ a system for splitting the exciting beam, as described for example in European patent applications 174 722 and 241 268.
The invention therefore relates to a method of measuring the luminescence emitted in a luminescent assay, which comprises employing at least one luminescent tracer compound and a luminescent compound used as an internal reference, which, when exposed to the same excitation wavelength, are capable of emitting at different wavelengths, xcex2 and xcex1 respectively, either by direct luminescence or by the induction of a luminescent emission, the luminescence of the reference compound reflecting the optical quality of the medium, and correcting the measurement of the luminescence emitted by the tracer compound at wavelength xcex2 on the basis of the measurement of the luminescence emitted by the reference compound at wavelength xcex1.
xe2x80x9cReference compoundxe2x80x9d is understood as meaning either a luminescent molecule emitting a direct luminescence, or a luminescent molecule capable of inducing a luminescent emission, said emission of a direct or indirect luminescence not being perturbed by the reagent system of the assay.
The luminescent compounds which can be used in the method of measurement according to the invention can emit directly either at their emission wavelength or at another wavelength, as for example in the case of a spectral shift associated with the reagent system of the assay.
If appropriate, the luminescent compounds which can be used in the method of measurement according to the invention can emit indirectly by inducing a luminescent emission, especially as in the case of homogeneous energy transfer methods.
Advantageously, the luminescent emissions at wavelengths xcex2 and xcex1 are detected simultaneously.
The method according to the invention, which uses a single exciting beam, permits easy and reliable measurement of the luminescence emitted in a luminescent assay without the need for complex equipment, by eliminating the perturbations due to the spectroscopic properties of the assay medium.
Advantageously, the emission wavelengths of the luminescent tracer compound and luminescent reference compound, xcex2 and xcex1, will be different but preferably similar (the difference being less than or equal to 100 nm, for example) so that the perturbation of the luminescent emission which is due to the absorption of the medium is produced in the same manner as regards the emission of the tracer compound and that of the reference compound.
It is pointed out that, advantageously, the method according to the present invention does not require the assay sample to be placed in a special measuring cell.
The luminescent emission of the reference compound at wavelength xcex1 enables the measurement made at wavelength xcex2 to be corrected. The correction can be effected for example by dividing the latter measurement by the measurement made at wavelength xcex1 Other means of correction can be used, for example a correction method, built into the apparatus, which consists in fixing a counting rate on the channel measuring the luminescent emission of the reference compound at wavelength xcex1. When this rate is reached, the end of the measurement on the channel measuring the luminescent emission at wavelength xcex2 is triggered. The value obtained on this channel is thereby corrected directly.
Other correction methods known to those skilled in the art can also be used.
According to a preferred feature, the invention relates to a method of measuring the fluorescence emitted in a fluorometric assay, which comprises employing at least one fluorescent tracer compound and a fluorescent compound used as an internal reference, which, when exposed to the same excitation wavelength, are capable of emitting at different wavelengths, xcex2 and xcex1 respectively, either by direct fluorescence or by the induction of a fluorescent emission, the fluorescence of the reference compound reflecting the optical quality of the medium, and the measurement of the fluorescence emitted by the tracer compound then being corrected on the basis of the measurement of the fluorescence emitted by the reference compound.
The method according to the invention makes it possible to achieve a measuring sensitivity of the order of picomol/liter, whereas the phenomena referred to above usually limit the sensitivity of the assay, especially in the case of homogeneous assays in a serum medium, to analyte concentrations of the order of micromol/liter.
According to an advantageous feature of the invention, the luminescent tracer compound and the reference compound are one and the same compound.
This first variant of the method according to the invention preferably applies when using a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, where the measurement of the emitted luminescence representing the quantity of analyte in the medium is made at a different emission wavelength from that of the tracer compound.
This case arises for example when the emitted luminescence representing the analyte results from an energy transfer between a luminescent donor compound and a luminescent acceptor compound, the latter emitting at a wavelength xcex2 and the donor compound, which also serves as the reference compound, emitting at a wavelength xcex1.
In particular, this case also arises when the tracer compound emits at different wavelengths xcex1 and xcex2 according to whether it does not or, respectively, does form part of the reagent system of the assay.
xe2x80x9cHomogeneous methodxe2x80x9d is understood as meaning an assay method in which the measurement does not require the prior separation of the constituents of the assay.
Surprisingly, it has in fact been found that the intensity of the signal emitted by the luminescent reference compound at wavelength xcex1 is practically constant. The emitted signal is therefore a function only of the optical properties of the medium in which the assay is performed, and not of the quantity of analyte, and can serve as a reference.
In the case of a luminescent emission resulting from an energy transfer, the signal reflecting both the quantity of analyte to be assayed and the optical properties of the measuring medium is detected at a wavelength xcex2 and corrected on the basis of the measurement made at wavelength xcex1.
Preferably, this first variant of the method according to the invention will be used in a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, with the aid of an excess method consisting in:
1) adding to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent donor compound,
2) adding a second reagent made up of one or more other receptors for said analyte, said second reagent being coupled with a luminescent acceptor compound,
3) incubating said medium after the addition of each reagent or after the addition of both reagents,
4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and
5) measuring the signal of the luminescent donor compound at a wavelength xcex1 this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength xcex2.
According to an advantageous feature, the first and second reagents used in the above-indicated luminescent methods of detecting and/or determining an analyte are added simultaneously to the medium containing the target analyte.
According to another feature of the invention, this variant of the method can be used in a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, with the aid of a competitive method consisting in:
1) adding, to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent donor compound,
2) adding a second reagent made up of the analyte coupled with a luminescent acceptor compound,
3) incubating said medium after the addition of each reagent or after the addition of both reagents,
4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and
5) measuring the signal of the luminescent donor compound at a wavelength xcex1 this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength xcex2.
This variant of the method can also advantageously be used in a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, with the aid of a competitive method consisting in:
1) adding, to said medium containing the target analyte, a first reagent made up of at least one receptor for said analyte, coupled with a luminescent acceptor compound,
2) adding a second reagent made up of the analyte coupled with a luminescent donor compound,
3) incubating said medium after the addition of each reagent or after the addition of both reagents,
4) exciting the resulting medium at the excitation wavelength of the luminescent donor compound, and
5) measuring the signal of the luminescent donor compound at a wavelength xcex1, this measurement serving as a reference, and the signal resulting from the energy transfer at a different wavelength xcex2.
In another advantageous use of the method of the invention, at least one of the receptors for the analyte is bound to a solid support.
According to another advantageous feature, the luminescent tracer compound and the reference compound are 2 different compounds excitable at the same wavelength, the reference compound emitting at a different wavelength from that used to measure the quantity of analyte.
This second variant of the method of the invention is preferred in particular in the use of a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, said method using a reagent coupled with a luminescent tracer compound and a reagent coupled with a heavy atom, or moieties containing a heavy atom, capable of modulating the signal of the luminescent tracer compound. An assay method of this type is described in European patent application 232 348.
In this case, in addition to the luminescent tracer compound coupled with the receptor for the analyte and emitting at a wavelength xcex2, another, free luminescent compound, serving as a reference and emitting at a wavelength xcex1, will be used whose signal is not modulated by the heavy atom effect and reflects the optical properties of the measuring medium, these 2 compounds being excited at the same wavelength.
Advantageously, this second variant of the method according to the invention is used in a homogeneous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, consisting in:
1) adding to said medium a first reagent made up of a receptor for said analyte,
2) adding a second reagent selected from the analyte or at least one of its receptors, one of the two reagents being coupled with a luminescent tracer compound and the other reagent containing a heavy atom or moieties containing a heavy atom, as well as a luminescent compound serving as an internal reference,
3) incubating the resulting medium either after the addition of each reagent or after the addition of both reagents,
4) exciting the resulting medium, and
5) measuring on the one hand the signal emitted by the luminescent tracer compound, said signal being modulated by the heavy atom effect at a wavelength xcex2, and on the other hand the signal emitted by the reference compound at a wavelength xcex1.
This second variant of the method, in which the luminescent tracer compound and the reference compound are two different compounds, can also advantageously be used in homogeneous methods of detecting and/or determining an analyte in a medium in which it may be present, with the aid of excess or competitive methods involving a donor/acceptor system, such as those described above for the first variant of the method of the invention.
Nevertheless, in the case of the second variant, a luminescent compound serving as an internal reference should be added during one of the reagent addition steps.
The signal of the luminescent reference compound will then be measured at a wavelength xcex1 and the signal resulting from the energy transfer will be measured at a wavelength xcex2, it then being possible for this measurement to be corrected on the basis of the measurement made at xcex1.
In the present description:
xe2x80x9canalytexe2x80x9d defines any substance or group of analogous substances to be detected and/or determined;
xe2x80x9creceptorxe2x80x9d defines any substance capable of binding specifically to a site on said analyte;
xe2x80x9cheavy atomxe2x80x9d defines an atom of high atomic number whose presence in the proximity of a fluorescent molecule is capable of inducing an increase in the spin-orbit coupling of the latter; examples which may be mentioned of appropriate heavy atoms are especially halogen atoms, mercury, thallium, lead and silver;
xe2x80x9cmoiety containing at least one heavy atomxe2x80x9d defines any chemical substance which naturally contains at least one heavy atom or to which at least one heavy atom can be bound.
According to a preferred feature, the measuring medium is a biological medium such as a serum medium.
Fluorescent compounds will advantageously be used as luminescent reference compounds and/or luminescent compounds usable as tracer compounds.
In particular, a fluorescent compound such as a rare earth chelate or cryptate will advantageously be used, especially a terbium, europium, dysprosium, samarium or neodymium chelate or cryptate. A terbium or europium cryptate will preferably be used.
In the fluorescent methods of detection and/or determination using the method of measurement of the invention, a rare earth cryptate described in European patent applications 180 492 and 321 353 will advantageously be chosen.
The terbium cryptate Tb trisbipyridine or the europium cryptate Eu trisbipyridine, as described in European patent application 180 492, or the cryptates Eu trisbipyridinediamine and Tb trisbipyridinediamine, described in European patent application 321 353, will preferably be used.
According to an advantageous feature, the fluorescent donor compound is a europium cryptate and the fluorescent acceptor compound is selected from allophycocyanin, allophycocyanin B, phycocyanin C or phycocyanin R.
It is also possible to use a phosphorescent compound, such as eosin or erythrosin, as the luminescent reference compound or donor tracer compound. In this case, it will be advantageous to use a fluorescent acceptor compound selected from chlorophylls such as those mentioned in European patent applications 71 991 and 314 406, or porphyrins such as those mentioned in European patent application 71 991, or else phthalocyanins such as those of international patent application WO 88 04777.
In the case of an assay in a liquid medium using phosphorescent donor compounds, the result will be read either from a solid support or by adding oxygen-capturing molecules to the measuring medium, these techniques being known to those skilled in the art.
Chlorophylls and phthalocyanins can also be used as fluorescent acceptor compounds, a europium cryptate or chelate being used as the donor compound.
According to a preferred feature, the luminescent tracer compound and/or the luminescent reference compound have a lifetime of more than one microsecond.
A modulatable light source, such as those described in Lakowicz, Principles of fluorescent spectroscopy, Plenum Press, New York, 1983, pp. 96-100, will advantageously be used as the light source permitting the excitation of the luminescent tracer and reference compounds.
The method of measurement of the invention has an important application in particular in fluorescent immunoassays, in both the so-called competitive and excess assay methods, which have been described in the prior art (Landon, Ann. Clin. Biochem., 1981, 18, 253, and E. SOINI et al., Clin. Chem., 1979, 25, 353).
In particular, the method of measurement of the invention can advantageously be used in immunoassays in a serum medium.
A further feature of the invention relates to a device for carrying out the method of measurement according to the invention.
This device comprises an exciting light source, means for collecting the light beam emitted following said excitation, and means for measuring the luminescence at two different wavelengths.
Advantageously, this device also comprises means for splitting the beam emitted following excitation.
Such a device is illustrated by way of example in FIG. 1.
FIG. 1 is a schematic illustration of a system for practicing the invention;
FIG. 2 is a graph in which the abscissa shows the prolactin concentration determine by the ELSA-PRL test in UI/ml and ordinates shows the concentration determine by a corrected measurement; and
FIG. 3 is a graph similar to the graph illustrated in FIG. 2 in which the abscissa is the same as FIG. 2 and the ordinate shows an uncorrected measurement.